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M D Houslay

Publications and source records attributed to M D Houslay.

At least 181 records · Page 10Linked to original sources
PubMed1986

Effects of delta 9-tetrahydrocannabinol on glucagon receptor coupling to adenylate cyclase in rat liver plasma membranes.

Delta-Tetrahydrocannabinol (delta 9-THC), the principal psychoactive constituent of Cannabis sativa, was found to increase glucagon activation of liver plasma membrane adenylate cyclase. In the presence of 30 microM delta 9-THC, the EC50 for glucagon was decreased by 60% from 7.6nM to 3.1 nM. 11-OH-delta 9-THC, a psychoactive metabolite of delta 9-THC, also increased glucagon activation of adenylate cyclase while two cannabinoids without marihuana-like psychoactive potency, cannabinol and cannabidiol, did not. At 30 microM, delta 9-THC either slightly decreased or had no effect on the activation of adenylate cyclase by GTP, Gpp(NH)p, fluoride ion, forskolin or ATP alone. Delta 9-THC had no effect on the binding of [125I] glucagon to liver plasma membranes. Arrhenius plots demonstrated that delta 9-THC and 11-OH-delta 9-THC, but not CBD, decreased the activation energy above the break temperature. Therefore, delta 9-THC increased the coupling of the glucagon receptor to adenylate cyclase apparently by removing a constraint on receptor-Ns coupling.

Adenylyl Cyclases↗
PubMed1986

Thrombin, unlike vasopressin, appears to stimulate two distinct guanine nucleotide regulatory proteins in human platelets.

The thrombin-stimulated GTPase activity of human platelets was additive with respect to the GTPase stimulation effected by prostaglandin E1, but not with that stimulated by adrenaline, vasopressin and platelet-activating factor (PAF). Treatment of platelet membranes with pertussis toxin partially inhibited the thrombin-stimulated GTPase, but had no effect on the vasopressin-stimulated GTPase activity, whereas cholera toxin treatment had no effect on either of these stimulated GTPase activities. Thrombin, adrenaline and PAF, but not vasopressin, inhibited the adenylate cyclase activity of isolated plasma membranes through the action of Ni only, this being inhibited by pertussis toxin. It is suggested that thrombin exerts effects through both the inhibitory guanine nucleotide regulatory protein Ni and through the putative guanine nucleotide regulatory protein, Np, involved in regulating receptor-stimulated inositol phospholipid metabolism. However, vasopressin appears to exert its effects solely through the putative Np.

ADP-Ribosylation Factors↗
PubMed1986

The action of islet activating protein (pertussis toxin) on insulin's ability to inhibit adenylate cyclase and activate cyclic AMP phosphodiesterases in hepatocytes.

Treatment of hepatocytes with islet activating protein (pertussis toxin) from Bordetella pertussis blocked the ability of insulin to inhibit adenylate cyclase activity both in broken plasma membranes and in intact hepatocytes. Such treatment of intact hepatocytes with pertussis toxin did not prevent insulin from activating the peripheral plasma membrane cyclic AMP phosphodiesterase although it did inhibit the ability of insulin to activate the 'dense-vesicle' cyclic AMP phosphodiesterase. The ability of glucagon pretreatment of hepatocytes to block insulin's activation of the plasma membrane cyclic AMP phosphodiesterase was abolished in pertussis toxin-treated hepatocytes. It is suggested that the ability of insulin to manipulate cyclic AMP concentrations by inhibiting adenylate cyclase and activating the plasma membrane and 'dense-vesicle' cyclic AMP phosphodiesterases involves interactions with the guanine nucleotide regulatory protein system occurring in liver plasma membranes.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Acidic phospholipid species inhibit adenylate cyclase activity in rat liver plasma membranes.

Incubation of rat liver plasma membranes with liposomes of dioleoyl phosphatidic acid (dioleoyl-PA) led to an inhibition of adenylate cyclase activity which was more pronounced when fluoride-stimulated activity was followed than when glucagon-stimulated activity was followed. If Mn2+ (5 mM) replaced low (5 mM) [Mg2+] in adenylate cyclase assays, or if high (20 mM) [Mg2+] were employed, then the perceived inhibitory effect of phosphatidic acid was markedly reduced when the fluoride-stimulated activity was followed but was enhanced for the glucagon-stimulated activity. The inhibition of adenylate cyclase activity observed correlated with the association of dioleoyl-PA with the plasma membranes. Adenylate cyclase activity in dioleoyl-PA-treated membranes, however, responded differently to changes in [Mg2+] than did the enzyme in native liver plasma membranes. Benzyl alcohol, which increases membrane fluidity, had similar stimulatory effects on the fluoride- and glucagon-stimulated adenylate cyclase activities in both native and dioleoyl-PA-treated membranes. Incubation of the plasma membranes with phosphatidylserine also led to similar inhibitory effects on adenylate cyclase and responses to Mg2+. Arrhenius plots of both glucagon- and fluoride-stimulated adenylate cyclase activity were different in dioleoyl-PA-treated plasma membranes, compared with native membranes, with a new 'break' occurring at around 16 degrees C, indicating that dioleoyl-PA had become incorporated into the bilayer. E.s.r. analysis of dioleoyl-PA-treated plasma membranes with a nitroxide-labelled fatty acid spin probe identified a new lipid phase separation occurring at around 16 degrees C with also a lipid phase separation occurring at around 28 degrees C as in native liver plasma membranes. It is suggested that acidic phospholipids inhibit adenylate cyclase by virtue of a direct headgroup specific interaction and that this perturbation may be centred at the level of regulation of this enzyme by the stimulatory guanine nucleotide regulatory protein NS.

Adenylyl Cyclase Inhibitors↗
PubMed1986

Platelet activating factor and U44069 stimulate a GTPase activity in human platelets which is distinct from the guanine nucleotide regulatory proteins, Ns and Ni.

Platelet-activating factor (PAF, 2-acetyl-1-alkyl-sn-glycero-3-phosphocholine) and the stable thromboxane-receptor agonist U44069 (9 alpha, 11 beta-epoxymethanoprostaglandin H2) stimulated GTPase activity in platelet membranes in a dose-dependent fashion, yielding Ka values of 12 nM and 27 nM respectively. The degree of GTPase activation elicited by these agents was found to be additive with the GTPase activation due to either the stimulatory (Ns) or inhibitory (Ni) guanine nucleotide regulatory proteins when activated by prostaglandin E1 and adrenaline (+propranolol) respectively. Treatment of membranes with either cholera or pertussis toxins, which inhibited markedly the receptor-mediated stimulation of the GTPase activities of Ns and Ni respectively, had no or only a small effect, respectively, on the GTPase activity stimulated by PAF and U44069. It is suggested that PAF and U44069, which stimulate inositol phospholipid metabolism in platelets, exert actions through a guanine nucleotide regulatory protein which is distinct from Ns and Ni.

Blood Platelets↗
PubMed1986

Identification and characterization of both the cytosolic and particulate forms of cyclic GMP-stimulated cyclic AMP phosphodiesterase from rat liver.

Two enzymes displaying cyclic GMP-stimulated cyclic AMP phosphodiesterase activity were purified from rat liver to apparent homogeneity: a 'particulate enzyme' found as an integral membrane protein associated with the plasma membrane, and a 'soluble' enzyme found in the cytosol. The physical properties of these enzymes were very similar, being dimers of Mr 134,000, composed in each instance of two subunits of Mr = 66,000-67,000. Both enzymes showed similar kinetics for cyclic AMP hydrolysis. They are both high-affinity enzymes, with kinetic constants for the particulate enzyme of Km = 34 microM and Vmax. = 4.0 units/mg of protein and for the cytosolic enzyme Km = 40 microM and Vmax. = 4.8 units/mg of protein. In both instances hydrolysis of cyclic AMP appeared to show apparent positive co-operativity, with Hill coefficients (happ.) of 1.5 and 1.6 for the particulate and cytosolic enzymes respectively. However, in the presence of 2 microM-cyclic GMP, the hydrolysis of cyclic AMP obeyed Michaelis kinetics (happ. = 1) for both enzymes. The addition of micromolar concentrations of cyclic GMP had little effect on the Vmax. for cyclic AMP hydrolysis, but lowered the Km for cyclic AMP hydrolysis to around 20 microM in both cases. However, at low cyclic AMP substrate concentrations, cyclic GMP was a more potent activator of the particulate enzyme than was the soluble enzyme. The activity of these enzymes could be selectively inhibited by cis-16-palmitoleic acid and by arachidonic acid. In each instance, however, the hydrolysis of cyclic AMP became markedly more sensitive to such inhibition when low concentrations of cyclic GMP were present. Tryptic peptide maps of iodinated preparations of these two purified enzyme species showed that there was considerable homology between these two enzyme forms.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

The phorbol ester TPA prevents the expression of both glucagon desensitisation and the glucagon-mediated block of insulin stimulation of the peripheral plasma membrane cyclic AMP phosphodiesterase in rat hepatocytes.

The phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate) causes a dose-dependent inhibition of the glucagon-stimulated adenylate cyclase activity expressed in plasma membranes isolated from TPA-treated hepatocytes. However, no observable inhibitory effect of TPA on adenylate cyclase activity was observed in cells which had been exposed to glucagon for 5 min, prior to isolation, to desensitise adenylate cyclase. The degree of inhibition of adenylate cyclase elicited by both glucagon desensitisation and TPA treatment of hepatocytes was identical. Pre-treatment of hepatocytes with TPA was also found to prevent glucagon from blocking insulin's activation of the peripheral plasma membrane cyclic AMP phosphodiesterase in intact hepatocytes. TPA treatment also inhibited the ability of cholera toxin to activate the peripheral cyclic AMP phosphodiesterase in intact hepatocytes. It is suggested that in these particular instances TPA and glucagon elicit mutually exclusive processes rather than TPA mimicking glucagon desensitisation per se.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

Insulin inhibits the cholera-toxin-catalysed ribosylation of a Mr-25000 protein in rat liver plasma membranes.

A method is described for preparing a plasma-membrane fraction from hepatocytes by a rapid, gentle, Percoll fractionation procedure. Cholera toxin elicited the ribosylation of a number of proteins in these membranes, including the components of the stimulatory guanine nucleotide regulatory protein, Ns. Insulin, however, inhibited the ability of cholera toxin to ribosylate a protein of Mr 25 000. The action was decreased in membranes from cells that had been pre-treated with glucagon. Ribosylation of both the components of Ns and the Mr-25 000 species occurred in whole cells treated with cholera toxin, because membranes from such treated cells exhibited decreased labelling when incubated with [32P]NAD+ and activated cholera toxin. The labelling of proteins, including the Mr-25 000 species, with [32P]NAD+ and cholera toxin in the plasma membranes was decreased by an inhibitor of ribosylation. Azido-GTP photoaffinity labelling identified several high-affinity GTP-binding proteins, including one of Mr 25 000. Cholera toxin failed to ribosylate the Mr-25 000 protein in membranes from cells that had been pre-treated with the tumour-promoting agent 12-O-tetradecanoylphorbol 13-acetate (TPA). In membranes from such treated cells, insulin actually allowed cholera toxin to label this species. As TPA activates protein kinase C, it is possible that the Mr-25 000 protein, or a species that interacts with it, is a substrate for phosphorylation. These observations may offer an explanation for some of the perturbing effects that TPA exerts on insulin's action. It is suggested that the insulin receptor interacts with the guanine nucleotide regulatory protein system in the liver, and that the Mr-25 000 species may be a component of Nin, a specific guanine nucleotide regulatory protein that has been proposed to mediate certain of the actions of insulin on target cells [Houslay & Heyworth (1983) Trends Biochem. Sci. 8, 449-452].

Animals↗
PubMed1985

Adenylate cyclase and a fatty acid spin probe detect changes in plasma membrane lipid phase separations induced by dietary manipulation of the cholesterol:phospholipid ratio.

Rats fed with a cholesterol supplement to their diet exhibited an increase in their plasma membrane cholesterol phospholipid (C/P)-lipid molar ratio from 0.72 to 0.98, whereas those fed the hypocholesterolaemic drug clofibrate in their diet exhibited a decrease in this ratio to 0.62. The properties of these membranes were analysed with regard to ligand-stimulated adenylate cyclase activity and the mobility of a fatty acid spin probe which allowed lipid phase separations to be identified. Membranes with elevated C/P ratios exhibited two distinct lipid phase separations, one at around 36 degrees C that was attributed to the external half of the bilayer and one at around 22 degrees C which was attributed to the inner half of the bilayer. Membranes with lowered C/P ratios exhibited a single lipid phase separation occurring at around 21 degrees C which was attributed to the lipids of the inner half of the bilayer. These results were compared with those obtained by manipulation of C/P ratios in vitro using liposome-cholesterol exchange techniques. Dietary manipulation of the C/P ratio of plasma membranes in vivo led to alterations in the fold stimulation of adenylate cyclase by various stimulatory ligands.

Adenylyl Cyclases↗
PubMed1984

N6-(Phenylisopropyl)adenosine prevents glucagon both blocking insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase and uncoupling hormonal stimulation of adenylate cyclase activity in hepatocytes.

Glucagon (10nM) prevented insulin (10nM) from activating the plasma-membrane cyclic AMP phosphodiesterase. This effect of glucagon was abolished by either PIA [N6-(phenylisopropyl)adenosine] (100nM) or adenosine (10 microM). Neither PIA nor adenosine exerted any effect on the plasma-membrane cyclic AMP phosphodiesterase activity either alone or in combination with glucagon. Furthermore, PIA and adenosine did not potentiate the action of insulin in activating this enzyme. 2-Deoxy-adenosine (10 microM) was ineffective in mimicking the action of adenosine. The effect of PIA in preventing the blockade by glucagon of insulin's action was inhibited by low concentrations of theophylline. Half-maximal effects of PIA were elicited at around 6nM-PIA. It is suggested that adenosine is exerting its effects on this system through an R-type receptor. This receptor does not appear to be directly coupled to adenylate cyclase, however, as PIA did not affect either the activity of adenylate cyclase or intracellular cyclic AMP concentrations. Insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase, in the presence of both glucagon and PIA, was augmented by increasing intracellular cyclic AMP concentrations with either dibutyryl cyclic AMP or the cyclic AMP phosphodiesterase inhibitor Ro-20-1724. PIA also inhibited the ability of glucagon to uncouple (desensitize) adenylate cyclase activity in intact hepatocytes. This occurred at a half-maximal concentration of around 3 microM-PIA. However, if insulin (10 nM) was also present in the incubation medium, PIA exerted its action at a much lower concentration, with a half-maximal effect occurring at around 4 nM.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

An assessment of the ability of insulin-stimulated cyclic AMP phosphodiesterase to decrease hepatocyte intracellular cyclic AMP concentrations.

Treatment of hepatocytes with either NH4Cl (10mM) or fructose (10mM) blocks insulin's activation of the 'dense-vesicle' cyclic AMP phosphodiesterase. The ability of insulin (10 nM) to decrease intracellular cyclic AMP concentrations raised by glucagon (10 nM) was unaffected by pre-treatment with either NH4Cl (10 mM) or fructose (10 mM). It is concluded that the 'dense-vesicle' enzyme does not play a significant role in this action of insulin and that as yet unidentified cyclic AMP phosphodiesterase(s) must be activated by insulin. Treatment of hepatocytes with either NH4Cl or fructose appeared to increase, reversibly, cyclic AMP phosphodiesterase activity. When N6-(phenylisopropyl)adenosine was used to prevent glucagon from blocking insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase activity, insulin's ability to decrease intracellular cyclic AMP concentrations in glucagon-treated hepatocytes was increased markedly. Insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase activity can exert a potent effect in decreasing intracellular cyclic AMP concentrations elevated by glucagon.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

Islet-activating protein blocks glucagon desensitization in intact hepatocytes.

Treatment of intact hepatocytes with islet-activating protein, from Bordatella pertussis, led to a pronounced increase in the ability of glucagon to raise intracellular cyclic AMP concentrations. Islet-activating protein, however, caused no apparent increase in the intracellular concentration of cyclic AMP under basal conditions. These effects were attributed to an enhanced ability of adenylate cyclase, in membranes from hepatocytes treated with islet-activating protein, to be stimulated by glucagon. When forskolin was used to amplify the basal adenylate cyclase activity, elevated GTP concentrations were shown to inhibit adenylate cyclase activity in membranes from control hepatocytes. This inhibitory effect of GTP was abolished if the hepatocytes had been pre-treated with islet activating protein. In isolated liver plasma membranes, islet-activating protein caused the NAD-dependent ribosylation of a Mr-40000 protein, the putative inhibitory guanine nucleotide regulatory protein, Ni. This effect was inhibited if guanosine 5'-[beta-thio]diphosphate rather than GTP was present in the ribosylation incubations. The ability of glucagon to uncouple or desensitize the activity of adenylate cyclase in intact hepatocytes was also blocked by pre-treating hepatocytes with islet-activating protein. Islet-activating protein thus heightens the response of hepatocytes to the stimulatory hormone glucagon. It achieves this by both inhibiting the expression of desensitization and also removing a residual inhibitory input expressed in the presence of glucagon.

Adenylate Cyclase Toxin↗
PubMed1984

On the possible role of biliverdin stimulation of cyclic AMP levels as a trigger for liver regeneration in the rat.

Contrary to a recent report by Okazaki et al. (Biochem. biophys. Res. Commun., 1978, 81, 512-520) we show that high concentrations of biliverdin (100 mg/kg, i.p.) slightly reduce the mitotic rate in rat liver. Adenylate cyclase and both the "high Km" and "low Km" phosphodiesterase of rat liver plasma membranes are inhibited by high concentrations of biliverdin. Biliverdin has no effect on cyclic AMP production in isolated hepatocytes. An intracellular binding protein (glutathione-S-transferase B) is shown to bind biliverdin providing a mechanism for maintaining a low free intracellular concentration of the tetrapyrrole analogous to that of albumin in plasma. In conclusion, these results are not consistent with a role for biliverdin in stimulating liver regeneration in the rat via a mechanism involving elevated cyclic AMP levels.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

Dimethylnitrosamine inhibits the glucagon-stimulated adenylate cyclase activity of rat liver plasma membranes and decreases plasma membrane fluidity.

The effect of the hepatocarcinogen dimethylnitrosamine on rat liver plasma membrane adenylate cyclase activity and lipid fluidity was assessed. Glucagon-stimulated adenylate cyclase activity exhibited a complex response to increasing concentrations of dimethylnitrosamine, whereas fluoride-stimulated adenylate cyclase activity was progressively inhibited. Maximal inhibitory effects were observed at a concentration of 15 mM in both cases. The activity of detergent-solubilized adenylate cyclase was unaffected by dimethylnitrosamine. ESR analysis using a fatty acid spin probe showed that dimethylnitrosamine produced a marked, dose-dependent reduction in the fluidity of the plasma membrane with a maximal effect occurring at 20 mM. Dimethylnitrosamine also elevated the temperature at which the lipid phase separation occurred in rat liver plasma membranes, from 28 degrees C to 31 degrees C. The non-carcinogenic but structurally similar compound, dimethylamine hydrochloride neither inhibited adenylate cyclase nor decreased plasma membrane fluidity. It is suggested that the decrease in membrane fluidity, induced by dimethylnitrosamine, via its effects on membrane fluidity, could influence plasma membrane function and cellular regulation.

Adenylyl Cyclases↗
PubMed1984

The phorbol ester, TPA inhibits glucagon-stimulated adenylate cyclase activity.

The ability of glucagon (10 nM) to increase hepatocyte intracellular cyclic AMP concentrations was reduced markedly by the tumour-promoting phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate). The half-maximal inhibitory effect occurred at 0.14 ng/ml TPA. This action occurred in the presence of the cyclic AMP phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) indicating that TPA inhibited glucagon-stimulated adenylate cyclase activity. TPA did not affect either the binding of glucagon to its receptor or ATP concentrations within the cell. TPA did inhibit the increase in intracellular cyclic AMP initiated by the action of cholera toxin (1 microgram/ml) under conditions where phosphodiesterase activity was blocked. TPA did not inhibit glucagon-stimulated adenylate cyclase activity in a broken plasma membrane preparation unless Ca2+, phosphatidylserine and ATP were also present. It is suggested that TPA exerts its inhibitory effect on adenylate cyclase through the action of protein kinase C. This action is presumed to be exerted at the point of regulation of adenylate cyclase by guanine nucleotides.

Adenylyl Cyclases↗